Figure 8. Role of glutaminase activity in cellular transformation.
(A) Mitochondrial fractions were prepared from equivalent numbers of control NIH 3T3 cells and cells stably expressing Dbl, Cdc42(F28L), Rac(F28L), or RhoC(F30L) that had been treated with 10 μM 968 or untreated. Top: Relative amounts of KGA in the different mitochondrial preparations as assessed using an antibody that recognizes both KGA isoforms. Bottom: The different mitochondrial fractions were assayed for basal glutaminase activity. 100% = 680 nM glutamine hydrolyzed per minute per 105 cells. Data are the average of 3 experiments (± s.d.).
(B) Mitochondrial fractions were prepared from equivalent numbers of the indicated breast cancer cells that had been treated with 10 μM 968 or untreated. Top: Relative amounts of KGA and VDAC in the mitochondrial preparations. Bottom: The different mitochondrial fractions were assayed for basal glutaminase activity. 100% = 750 nM glutamine hydrolyzed per minute per 105 cells. Data represent the average of 3 experiments (± s.d.).
(C) Mitochondrial fractions from NIH 3T3 cells stably expressing Dbl that were cultured for 2 days in the presence or absence of 2 μM BAY 11-7082, or transfected with control siRNA or siRNAs targeting p65/RelA. Top: Relative amounts of KGA in the mitochondria from the indicated cells as assessed using an antibody that recognizes both KGA isoforms, and relative efficiencies of two siRNAs targeting p65/RelA. Bottom: The basal glutaminase activity for the different mitochondrial fractions. 100% represents the activity for untreated Dbl-transformed cells. Data represent the average of 2 experiments (± range).
(D) Mitochondrial fractions were prepared from HMECs, and SKBR3 cells that had been treated with 2 μM BAY 11-7082 or untreated, or transfected with siRNAs targeting p65/RelA. Top: Relative amounts of KGA in the different mitochondrial fractions as assessed using an antibody that reorganizes both KGA isoforms, and relative efficiencies of two siRNAs targeting p65/RelA. Bottom: The basal glutaminase activity for the different mitochondrial fractions. 100% represents the activity for untreated SKBR3 cells. Data represent the average of 2 experiments (± range).
(E) Top: V5-GAC was transiently expressed in control NIH 3T3 cells or NIH 3T3 cells stably expressing Dbl that were treated with either 2 μM BAY 11-7082, 10 μM 968, or untreated, and then immunoprecipitated from the different samples. Bottom: Basal activity for V5-GAC immunoprecipitated from the different cells. Data represent the average of 3 experiments (± s.d.).
(F) V5-GAC was transiently expressed in NIH 3T3 cells stably expressing Dbl. The V5-GAC was immunoprecipitated, treated with alkaline phosphatase for 1 hour at 37°C (or untreated), and assayed for activity in the presence and absence of phosphate.